单色光照射对体外培养的Müller细胞生长及细胞因子表达的影响

摘要

背景 530 nm单色光照射可诱导动物产生近视.Müller细胞作为视网膜上主要的胶质细胞,参与近视的形成,但Müller细胞在单色光诱导的近视形成过程中的具体作用尚不清楚. 目的 研究530 nm单色光照射对体外培养的大鼠视网膜Müller细胞的生物学特征及近视相关细胞因子表达的影响,探讨Müller细胞在单色光诱导的近视形成中的作用.方法 在自制光照度分别为125、250、500 lx波长530 nm单色光设备的细胞培养箱中用常规细胞培养基培养永生化大鼠Müller细胞,另用无光照的常规培养法培养细胞作为对照组.细胞培养6、12、24 h后,用四甲基偶氮噻唑蓝(MTT)法检测细胞在波长570 nm处的吸光度(A570)值；培养48 h后用流式细胞仪检测各组大鼠Müller细胞的细胞周期,比较各组G2期与G1期细胞数的比值；采用逆转录PCR(RT-PCR)法检测各组大鼠Müller细胞中转化生长因子-β1(TGF-β1)、酪氨酸羟化酶(TH)、诱导型一氧化氮合成酶(iNOS)和碱性成纤维细胞生长因子(bFGF) mRNA表达量的改变.采用两因素方差分析比较各组间大鼠Müller细胞随不同培养时间的变化增生值的差异,采用单因素方差分析比较各组间不同细胞周期细胞数的变化以及大鼠Müller细胞中各近视相关因子mRNA表达量的差异. 结果 530 nm单色光照射后,Müller细胞均呈典型的多角形,细胞大小均匀,边界清晰.与对照组比较,125 lx组、250 lx组大鼠Müller细胞在培养0、6、12、24 h时A570值的差异均无统计学意义(P＞0.05),但500 lx组光照12h、24 h后细胞A570值明显低于对照组,差异均有统计学意义(P=0.013、0.001).与对照组比较,125 lx组、250 lx组大鼠Müller细胞G2/G1细胞数比值的差异均无统计学意义(P=0.073、0.330),500 lx组G2/G1细胞数比值明显降低,与对照组、250 lx组比较差异均有统计学意义(P=0.028、0.038).RT-PCR检测结果显示,250 lx组TGF-β1 mRNA的表达明显高于500 lx组(P=0.006)；125 lx组、250 lx组iNOS mRNA表达依次上调,250 lx组与对照组比较差异有统计学意义(P=0.001),而500 lx组表达下调,与对照组比较差异有统计学意义(P=0.000).与对照组比较,125 lx组、250 lx组bFGF mRNA的表达量均上调,但500 lx组细胞中bFGF mRNA表达量下调,差异均有统计学意义(P=0.002、0.000、0.005).与对照组比较,250 lx组细胞中TH mRNA的表达量明显增加,500 lx组细胞中TH mRNA表达量明显下调,差异均有统计学意义(P=0.000、0.001). 结论 光照度＞250 lx的530 nm单色光照射可以通过抑制大鼠Müller细胞的生长,并下调细胞中近视相关细胞因子的表达而在近视的形成中发挥作用.%Background Research showed that exposure of 530 nm monochromatic light can induce myopia in animal,and retinal Müller cells participate in the formation of myopia.However,the effect and mechanism of retinal Müller cells during the formation of monochromatic light induced-myopia is below understood.Objective This study was to investigate biologic characteristics of rat retina Müller cells and the expression of cell factors in Müller cells after being illuminated by the 530 nm monochromatic light,and discuss the role of the retina Müller cells in myopia induced by monochromatic light.Methods Immortalized rat retinal Müller cells were cultured with DMEM containing 10％ fetal bovine serum in a self-made cell incubator with monochromatic light by adjusting luminance of 530 nm LED source.The cells were exposed to 125,250 and 500 lx luminance respectively for 6,12 and 24 hours,and the cells without light-irradiation were used as control.The growth of the cells under the different light time and different illuminations was described by MTT as the absorbance at the wavelength 570 nm (A570),and cell cycle analysis of Müller cells was performed by flow cytometry 48 hours after cultured,and the expression of transforming growth factor-beta 1 (TGF-β1),tyrosine hydroxylase(TH),inducible nitric oxide synthase(iNOS) and basic fibroblast growth factor(bFGF)in the cells were detected by reverse transcription PCR(RT-PCR),respectively.Results The Müller cells were uniform in size with polygonal shape and defined edges.No statistically significant difference was found in the A570 value in the cells of the 125 lx and 250 lx illuminated groups compared with the control group in various time points(P＞0.05).However,significant lowing was seen in the A570 value in the cells of the 500 lx illuminating for 12 hours and 24 hours in comparison with the control group (P =0.013,0.001).Compared with the control group,the ratio of the number between G2 and G1 phase was not significantly declined in 125 lx,250 lx illuminating for 48 hours (P =0.073,0.330),and the ratio in the 500 lx illuminating group was significantly lower than those in the 250 lx illuminated group and the control group (P =0.028,0.038).RT-PCR revealed that the expression of TGF-β1 mRNA in the cells was higher in the 250 lx illuminated group than that of the 500 lx illuminated group (P=0.006).The expression of iNOS mRNA was gradually upregulated in the 250 lx illuminated group compared with the control group (P =0.001),but that in the 500 lx illuminated group was downregulated (P =0.000).The expression of bFGF mRNA was raised in the 125 lx and 250 lx groups but reduced in the 500 lx group when compared with the control group(P=0.002,0.000,0.005).Also,the expression of TH mRNA was significantly increased in the 250 lx group(P=0.000),but decreased in the 500 lx group(P=0.000,P=0.001).Conclusions The monochromatic light of 530 nm can inhibit the growth of rat Müller cells and downregulate the expression of myopia-related cell factors and therefore exert effect in the formation of myopia.